Detection of Microorganisms With a Fluorescence-Based Device

ABSTRACT

A device and method for detecting by fluorescence microbial growth from sample substances are disclosed. For example, a method for the detection of visible-band fluorescence signals generated by at least one fluorescing compound excited by ultraviolet energy, comprising exciting said at least one fluorescing compound with ultraviolet energy emitted from a light-emitting diode comprising wavelengths below 400 nanometers, and detecting a visible-band fluorescence signal generated by said at least one excited fluorescing compound with at least one light detector sensitive to electromagnetic energy comprising wavelengths greater than or equal to 400 nanometers wavelength. For example, a device for detecting visible-band fluorescence signals generated by at least one fluorescing compound excited by ultraviolet energy, comprising at least one ultraviolet light-emitting diode generating electromagnetic radiation comprising wavelengths below 400 nanometers and capable of exciting the at least one fluorescing compound, at least one light detector sensitive to electromagnetic energy comprising wavelengths greater than or equal to 400 nanometers wavelength for the detection of visible-band fluorescence signals generated by the at least one fluorescing compound.

This application claims the benefit of U.S. Provisional Application No.60/592,166 filed Jul. 29, 2004, and International Application No.PCT/US05/04331, filed Feb. 11, 2005, which are incorporated herein byreference.

The present invention relates to fluorescence-based devices fordetecting microbial growth from test samples.

It is necessary to test various industrial substances, such as food,pharmaceuticals, cosmetics and water, for microbial contamination. Onearea of biological testing of food, dairy, pharmaceutical, cosmetic andrelated types of products involves the estimation of total numbers ofbacteria, yeasts and molds, as well as concentrations of specific groupsof organisms within the material. One widely used method is known as the“Standard Plate Count” method, and involves culturing a diluted sampleof the product in an agar growth medium. The plates containing thesample and the growth medium are incubated (e.g., 32° C.-40° C.) for 24hours to 5 days, depending upon the assay. After incubation, colonies ofmicroorganisms which have grown in the agar are counted.

Optical methods have been successfully used to classify microorganismsin clinical samples (e.g., PASCO by Difco, Detroit, Mich.). Although itwould be desirable to utilize a colorimetric method, or any otheroptical method, for detecting microbial growth in industrial samples,the solid substances of the test samples disposed in an aqueous mediausually cause optical interference for a detection system. Morespecifically, when solid substances are disposed in a media to allow forculturing microorganisms, the colorimetric detection system must passlight either through or reflect light from the media containing thesolid substance. In most of the cases, the solid substances interferewith the spectral characteristics of the media, yielding a poorsignal-to-noise ratio of the detection system.

A device for continuously monitoring the biological activity in aspecimen is described by Eden in U.S. Pat. No. 5,366,873. It describes adevice and method for detecting microbial growth from a samplesubstance. The device includes a container which is at least partiallytransparent and fluid disposed in the container for cultivatingmicroorganisms therein. An indicator substance is disposed in the fluidlayer for undergoing transformation in the presence of microorganismgrowth. A second layer, composed of semi fluid substance, indicators andother substances, such as growth media, is disposed in the container.The substances within the semi-fluid phase are in equilibrium with thesubstances in the fluid layer and provide a barrier to solid substancesintroduced into the fluid layer while providing a zone within whichchanges in the indicator substance, due to microbial growth, can bedetected. In practice, the indicator substance has been dyes that areaffected by the PH variations in the fluid layer.

The present invention extends the scope of the above patent by employingfluorescing indicator substances to enhance the measurement sensitivityand the group of detected microorganisms. In certain embodiments of thepresent invention, there is provided a device and method for detectingmicrobial growth from a sample substance. The device can comprise atleast one container which is at least partially transparent toelectromagnetic radiation in the visual and/or the ultravioletwavelength ranges and fluid disposed in the at least one container forcultivating microorganisms therein. At least one fluorescing indicatorsubstance can be disposed in the fluid layer for undergoingtransformation in the presence of microorganism growth. A second layer,composed of at least one semi-fluid substance, indicators and othersubstances, such as growth media, can be disposed in the container. Thesubstances within the semi-fluid phase can be in equilibrium with thesubstances in the fluid layer and can provide a barrier to solidsubstances introduced into the fluid layer while providing a zone withinwhich fluorescence changes in the indicator substance due to microbialgrowth can be detected.

One embodiment of the invention relates to a method for the detection ofvisible-band fluorescence signals generated by at least one fluorescingcompound excited by ultraviolet energy, comprising:

exciting the at least one fluorescing compound with ultraviolet energyemitted from a light-emitting diode comprising wavelengths below 400nanometers; and

detecting a visible-band fluorescence signal generated by the at leastone excited fluorescing compound with at least one light detectorsensitive to electromagnetic energy comprising wavelengths greater thanor equal to 400 nanometers.

Another embodiment of the invention relates to method for the detectionof visible-band fluorescence signals generated by at least onefluorescing compound excited by ultraviolet energy, comprising:

exciting the at least one fluorescing compound with ultraviolet energy;and

detecting a visible-band fluorescence signal generated by the at leastone excited fluorescing compound with a light detector sensitive toelectromagnetic energy comprising wavelengths greater than or equal to400 nanometers.

Yet another embodiment of the present invention relates to a device fordetecting visible-band fluorescence signals generated by at least onefluorescing compound excited by ultraviolet energy, comprising:

at least one ultraviolet light-emitting diode generating electromagneticradiation comprising wavelengths below 400 nanometers and capable ofexciting the at least one fluorescing compound; and

at least one light detector sensitive to electromagnetic energycomprising wavelengths greater than or equal to 400 nanometers for thedetection of visible-band fluorescence signals generated by the at leastone fluorescing compound.

A further embodiment of the present invention relates to a device fordetecting visible-band fluorescence signals and visible-band secondarysignals generated by at least one fluorescing compound excited byultraviolet and visible-band energy, comprising:

at least one ultraviolet light-emitting diode generating electromagneticradiation comprising wavelengths below 400 nanometers, the ultravioletlight-emitting diode capable of exciting the at least one fluorescingcompound, thereby generating the visible-band fluorescence signal;

at least one visible-band light-emitting diode generatingelectromagnetic radiation comprising wavelengths greater than or equalto 400 nanometers, the visible-band light-emitting diode capable ofinteracting with at least one visible dye compound, thereby generatingthe visible-band secondary signal; and

at least one light detector sensitive to electromagnetic energycomprising wavelengths greater than or equal to 400 nanometers fordetecting the visible-band fluorescence signal and the visible-bandsecondary signal.

Advantages of the present invention will be readily appreciated as thesame becomes better understood by reference to the following detaileddescription when considered in connection with the accompanying drawingswherein:

FIG. 1 shows one embodiment of the present invention; and

FIG. 2 shows another embodiment of the present invention.

Generally, the present invention provides a device for detectingmicrobial growth from a sample substance wherein the device includes atleast one container which is at least partially transparent to visualand/or ultraviolet (UV) radiation. A fluid layer can be disposed in thecontainer for cultivating microorganisms therein. A fluorescingindicator substance can be disposed in the fluid layer for undergoingtransformation in the presence of microorganism growth. A barrier layercan be disposed in the container which is a semi-fluid substance, thefluid portion of which is the same composition as the fluid layer inwhich the microorganisms are cultivated. Therefore, the fluid in thesemi-fluid layer is in equilibrium with the fluid layer. The semi-fluidsubstance provides a barrier to solid substances introduced into thefluid layer while providing a zone within which changes in the at leastone fluorescing indicator substance, due to microbial growth, can bedetected.

More specifically, the barrier layer can be comprised of gelling agents,such as agar. In carrying out the present invention, any type of gellingsubstance or agar, as defined in the Merck Index, can be utilized. Thereare several commercial gelling products available which are suitable,including gelatin, carrageenan and pectin.

The important property of such gelling agents used in the presentinvention is their ability to transfer ions, such as H+ and smallmolecules, while blocking out bacteria and larger debris particles. Ifthe concentration of the small particles changes due to organism growth(e.g., pH or Redox reactions), the concentration of the identicalparticles in the barrier layer will track those changes as well. Thediffusion coefficient of the barrier layer determines the rate in whichvariations in the liquid layer are tracked by identical changes in thebarrier layer.

FIG. 1 illustrates a typical configuration of the various components ofa system which can be utilized in accordance with this invention. Thevial 10 is made of UV-transparent material (e.g., glass, UV-transparentplastics). The barrier layer 16 may be composed of any available agar(e.g., Muller Hinton Agar by Difco, Detroit, Mich.) and non-toxicfluorescing dye 14, such as Umbelliferon. This layer can be manufacturedby dispensing the mixture, thermally sterilized, to the bottom of thevial 10 and letting it solidify at room temperature. A sterile mixtureof the liquid media 12 and dye 14 is poured at room temperature on topof the barrier layer.

The test sample 28 is placed in the fluid layer. The vial 10 is thenplaced in an incubating device, at an appropriate temperature, topromote growth of organisms. The incubating device can be an airincubator, heating and cooling blocks or heat exchanger.

An ultraviolet light source 18 is positioned at the bottom part of thevial 10 such that the transmitted UV light is directed through theUV-transparent walls of the vial 10 and the barrier layer 16. The lightsource can comprise any long or short wave ultraviolet from various UVsources. For example, greater than 50% of the wavelengths from the lightsource can be below 400 nanometers. In certain embodiments, at least 75%of the wavelengths from the light source can be below 400 nanometers,for example, at least 85% of the wavelengths from the light source canbe below 400 nanometers, and at least 95% of the wavelengths from thelight source can be below 400 nanometers.

Light emitting diodes (LED) can be used to provide the ultravioletlight. In embodiments of the present invention, greater than 50% of thelight generated by the light emitting diode can have a wavelength below400 nanometers, such as, for example, greater than 75%, greater than85%, and greater than 95%. In an embodiment of the invention, a longwavelength ultraviolet Light Emitting Diode (e.g., 350 to 400nanometers) can be utilized.

In another embodiment of the present invention, a multiplicity of lightemitting diodes can be controlled by the controller 20, which provideselectrical energy which can be spatially uniform and stable.

Suitable materials that can be used as the at least one fluorescingcompound include materials that emit visible light upon exposure toultraviolet radiation, such as, for example, umbelliferons andcoumarins. In dealing with fluorescing essays, one should remember thatthe wavelength of the radiation emitted from the fluorescing compound islonger than that of the light source. For example, radiatingumbelliferon with a UV light source of 380 nanometers (invisible)generates a blue-green visible radiation. Consequently, care should betaken that the light sensor will not be influenced by stray lightgenerated by the UV light source. If the UV source 18 is placed directlyfacing the light sensor 22, as shown in FIG. 1, an additional band passoptical filter 23 is required to block the influence of the UV radiationon the sensor. Alternatively, the UV light source 18 and the sensor 22can be placed next to each other facing the UV-transparent section ofthe vial at specific angles, as shown in FIG. 2, so that the fluorescingradiation is reflected back to the light sensor. Since the fluorescingradiation is equally radiated in all directions, the specific angles canbe set to minimize UV reflected light, thereby allowing the light sensorto measure only the fluorescing energy.

The dynamic changes of the fluorescing light, which is the indicator ofbacterial activity, is converted to electrical energy utilizing a lightsensor 22. Although a wide variety of sensors may be utilized (e.g.,photo voltaics, photodiodes, phototransistors, photo multipliers,charged coupled devices (CCD) and multi-channel devices) low-cost solidstate sensors can be employed due to the high energy of light reachingthe sensor. Therefore, each vial can have its own pair of light sourceand sensor, thus eliminating complex mechanical indexing devicesutilized in optical readers and thereby increasing the reliability andthe operating life of the instrument. The light emitting diode canprovide either stationary (constant) or pulsated energy. If anadditional light emitting diode operating in the visible range isemployed, one of the light emitting diodes can be driven at a constantlevel of energy while the other can be pulsated, allowing a single lightsensor to detect both signals. In another embodiment, both UV lightemitting diode and the visible-range light emitting diode can becombined in a single package forming dual-band UV and visible lightsources that can be independently activated.

In one embodiment of the invention, readings are taken every sixminutes, and the analog data can be converted by the converter 24 todigital form. The process data can be transferred to a processor 26,where it can be displayed, stored and analyzed for real time detection.

The gelling agent or agar can be positioned in the container such thatit can be in a transparent region of the container to facilitatemeasurement of changes in this phase of the system when in use. If thecontainer is a vial or tube, typically the agar could be placed at thebottom of such receptacle, as illustrated in FIG. 1, and would beapproximately 2 to 3 mm thick. The agar also could be in the form of adisc, attached to any wall of the container or other configuration asmay be convenient in accomplishing the measurement which is an object ofthe present invention.

The semi-fluid layer (e.g., the agar or gelling phase) can be situatedin the liquid phase within the container such that the liquid substanceswithin the agar are in equilibrium with the remaining liquid in thecontainer. In the practice of the present invention, the liquid phasewithin the container can be a liquid medium suitable for culturingmicroorganism growth. A sample of a substance which may harbormicroorganisms can be placed in the liquid phase in the container andincubated to promote growth of the microorganisms. When microorganismsare present, their growth will result in changes in the composition ofthe liquid phase throughout the container inasmuch as the liquid in thesemi-fluid or agar phase can be in equilibrium with the remainder of theliquid in the container. The contents of the liquid growth medium can beselected to result in a wide variety of changes in the liquidcomposition that can be detected and measured, as set forth in moredetail below. The change in the composition of the liquid growth mediumcan be detected and measured in the semi-fluid phase, which can be freeof the sample that can be being tested and free of microorganisms. Thesample being tested is usually too large molecularly to penetrate theagar phase, as are the microorganisms. Thus, the semi-fluid phaseprovides a zone within which changes in the liquid phase, brought on bymicroorganism growth, can be readily detected and measured without anyinterference from the test sample.

The liquid phase of the present invention can be a medium suitable forthe promotion of microorganism growth and for the maintenance of theviability of the microorganisms. Such growth media are well known in theart.

After a test sample has been placed in the liquid phase of thecontainer, the container can be incubated at an appropriate temperature(e.g., about 15° C. to 65° C.) for about 24 to 48 hours, or some othersuitable time period, after which changes in the at least onefluorescing substance can be measured. Changes in the at least onefluorescing substance are detected and measured in the semi-fluid phaseby analyzing the fluorescence changes related to microorganism growth.Changes in the indicator substance can be detected and measured in thesemi-fluid phase since the liquid in this phase can be in equilibriumwith the remaining liquid in the container. Thus, any changes whichoccur in the fluorescence substance will be present throughout thecontainer. Detection and measurement in the semi-fluid phase free oflarge molecules (e.g., the sample being tested) and microorganismsprovides an accurate and consistent means of detecting microorganismgrowth with a high signal-to-noise ratio.

The container used in the present invention can be glass or longUV-transparent plastics, such as polystyrenes. The entire container neednot be transparent, but the portion of the container surrounding thesemi-fluid phase must be transparent to permit measurement of any changein the indicator substance in response to microorganism growth. Also,the container can be any shape or size, but typically will be a vial ora tube which can be closed once the agar phase and liquid phase areincorporated therein. Once the two phases are loaded in the container,they can be shipped to the site needed for performing analysis of testsamples. No special temperature or storage requirements for thecontainer exist.

In an embodiment of the present invention, a multiplicity of fluorescingcompounds can be excited by a multiplicity of light emitting diodes inorder to cause the fluorescing compounds to emit visible light. Thefluorescing compounds can be present in the same container prior toexcitement with the light emitting diodes, or the fluorescing compoundscan be present in different containers prior to excitement. In certainembodiments, a single light detector or a multiplicity of lightdetectors can be used. In certain embodiments, a multiplicity ofcontainers are used, with each container having its own light emittingdiode and its own light detector.

In certain embodiments of the invention comprising at least oneultraviolet light emitting diode and at least one visible-band lightemitting diode, one of the light emitting diodes can be generatingstationary energy and the other light emitting diode can be pulsated,thereby generating a combination of constant energy and pulsated energydirected to the light detector and corresponding to the individualfluorescence signal and the secondary signal. The ultraviolet lightemitting diode and visible-band light emitting diode can be packaged ina single enclosure, thereby forming a dual band light emitting diode.

In one embodiment of the invention comprising at least one ultravioletlight emitting diode and at least one visible-band light emitting diode,one of the light emitting diodes can be activated for a specific amountof time while the other light emitting diode can be deactivated,followed by activating the deactivated light emitting diode anddeactivating the activated light emitting diode, thereby alternatelygenerating the fluorescence signal and the secondary signal atconsecutive periods of time.

In a further embodiment of the invention comprising at least oneultraviolet light emitting diode and at least one visible-band lightemitting diode, the interaction of the visible-band light emitting diodewith the at least one visible dye compound defines the opticaltransmittance of the at least one visible dye compound.

Suitable visible dyes compounds include, for example, pH indicators suchas Bromcresol Purple, Phenol Red, Bromcresol Green, Bromphenol Blue,Bromthymol Blue; and Redox indicators such as resazurin, methylene Blue,tetrazolium and thionine.

The invention has been described in an illustrative manner, and it is tobe understood that the terminology which has been used, is intended tobe in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. It is, therefore, to beunderstood that within the scope of the appended claims, the inventionmay be practiced otherwise than as specifically described.

1. A method for the detection of visible-band fluorescence signalsgenerated by at least one fluorescing compound excited by ultravioletenergy, comprising: exciting said at least one fluorescing compound withultraviolet energy emitted from a light-emitting diode comprisingwavelengths below 400 nanometers; and detecting a visible-bandfluorescence signal generated by said at least one excited fluorescingcompound with at least one light detector sensitive to electromagneticenergy comprising wavelengths greater than or equal to 400 nanometers.2. The method of claim 1, wherein said light detector is aphoto-transistor with diminished sensitivity below 400 nanometerswavelength.
 3. The method of claim 1, wherein said at least onelight-emitting diode generates a stationary level of energy.
 4. Themethod of claim 1, wherein said at least one light-emitting diodegenerates pulsated energy.
 5. The method of claim 1, wherein said atleast one said fluorescing compound is chosen from umbelliferons andcoumarins.
 6. The method of claim 1, wherein said at least onefluorescing compound is dissolved in liquid.
 7. The method of claim 1,wherein said at least one fluorescing compound is dissolved in agar. 8.The method of claim 1, wherein said at least one fluorescing compound isimpregnated in a matrix.
 9. The method of claim 6, wherein biologicalcells are grown in said liquid.
 10. The method of claim 9, wherein saidbiological cells are microorganisms.
 11. The method of claim 10, whereinsaid microorganisms cause said at least one visible dye compound to emita visible band secondary signal when exposed to visible light.
 12. Themethod of claim 1, wherein said at least one light emitting diode andsaid at least one light detector face each other.
 13. The method ofclaim 1, wherein said at least one light emitting diode and said atleast one light detector are arranged at an angle.
 14. The method ofclaim 13, wherein said at least one light detector detects no directlight generated by said at least one light emitting diode.
 15. Themethod of claim 1, wherein no band-pass filter is employed.
 16. Themethod of claim 1, wherein a multiplicity of fluorescing compounds areexcited by a multiplicity of light emitting diodes.
 17. The method ofclaim 16, wherein a multiplicity of containers are employed.
 18. Amethod for the detection of visible-band fluorescence signals generatedby at least one fluorescing compound excited by ultraviolet energy,comprising: exciting said at least one fluorescing compound withultraviolet energy; and detecting a visible-band fluorescence signalgenerated by said at least one excited fluorescing compound with a lightdetector sensitive to electromagnetic energy comprising wavelengthsgreater than or equal to 400 nanometers.
 19. A device for detectingvisible-band fluorescence signals generated by at least one fluorescingcompound excited by ultraviolet energy, comprising: at least oneultraviolet light-emitting diode generating electromagnetic radiationcomprising wavelengths below 400 nanometers and capable of exciting saidat least one fluorescing compound; and at least one light detectorsensitive to electromagnetic energy comprising wavelengths greater thanor equal to 400 nanometers for the detection of visible-bandfluorescence signals generated by said at least one fluorescingcompound.
 20. The device of claim 19, wherein said light detector is aphoto-transistor with diminished sensitivity below 400 nanometerswavelength.
 21. The device of claim 19, wherein said light-emittingdiode generates a stationary level of energy.
 22. The device of claim19, wherein said light-emitting diode generates pulsated energy.
 23. Thedevice of claim 19, wherein said at least one fluorescing compound ischosen from umbelliferons and coumarins.
 24. The device of claim 19,wherein said fluorescing compound is dissolved in liquid.
 25. The deviceof claim 19, wherein said fluorescing compound is dissolved in agar. 26.The device of claim 19, wherein said fluorescing compound is impregnatedin a matrix.
 27. The device of claim 24, wherein biological cells arecapable of being grown in said liquid.
 28. The device of claim 27,wherein said biological cells are microorganisms.
 29. The device ofclaim 28, wherein said microorganisms cause at least one visible dyecompound to emit a visible band secondary signal when exposed to visiblelight.
 30. The device of claim 19, wherein said at least one lightemitting diode and said at least one light detector face each other. 31.The device of claim 19, wherein said at least one light emitting diodeand said at least one light detector are arranged at an angle.
 32. Thedevice of claim 31, wherein said at least one light detector detects nodirect light generated by said at least one light emitting diode. 33.The device of claim 19, wherein a multiplicity of fluorescing compoundsare excited by a multiplicity of light emitting diodes.
 34. The deviceof claim 33, wherein a multiplicity of containers are employed.
 35. Thedevice of claim 19, wherein no band-pass filter is employed.
 36. Thedevice of claim 19, further comprising at least one band-pass filterlocated in the path of said electromagnetic radiation in front of alight sensitive area of said at least one light detector.
 37. Aninstrument for simultaneous measurements of a multiplicity offluorescing compounds comprising multiple units each comprising thedevice according to claim
 19. 38. A device for detecting visible-bandfluorescence signals and visible-band secondary signals generated by atleast one fluorescing compound excited by ultraviolet and visible-bandenergy, comprising: at least one ultraviolet light-emitting diodegenerating electromagnetic radiation comprising wavelengths below 400nanometers, said at least one ultraviolet light-emitting diode capableof exciting said at least one fluorescing compound, thereby generatingsaid visible-band fluorescence signal; at least one visible-bandlight-emitting diode generating electromagnetic radiation comprisingwavelengths greater than or equal to 400 nanometers, said at least onevisible-band light-emitting diode capable of interacting with at leastone visible dye compound, thereby generating said visible-band secondarysignal; and at least one light detector sensitive to electromagneticenergy comprising wavelengths greater than or equal to 400 nanometersfor detecting said visible-band fluorescence signal and saidvisible-band secondary signal.
 39. The device of claim 38, wherein oneof said light-emitting diodes is generating stationary'energy andanother light-emitting diode is pulsated.
 40. The device of claim 38,wherein one light-emitting diode is activated while anotherlight-emitting diode is deactivated, followed by activating saiddeactivated light-emitting diode and deactivating said activatedlight-emitting diode, thereby alternately generating said fluorescencesignal and said secondary signal.
 41. The device of claim 38, whereinsaid at least one ultraviolet light-emitting diode and said at least onevisible-band light-emitting diodes are packaged in a single enclosure.42. The device of claim 38, wherein the interaction of said visible-bandlight-emitting diode with said at least one visible dye compound definesthe optical transmittance of said at least one visible dye compound.